Osteogenic compositions containing a coloring agent

ABSTRACT

An osteogenic composition is provided for implantation at or near a target tissue site beneath the skin, the osteogenic composition comprising a growth factor and a coloring agent, wherein the coloring agent imparts color to the growth factor to allow the user to see the growth factor at or near the target tissue site. In some embodiments, a method is provided for accelerating bone repair, the method comprising mixing bone morphogenic protein- 2  and a coloring agent to form a mixture; applying the mixture to a surface of a porous collagen matrix, wherein the coloring agent allows the user to see bone morphogenic protein- 2  distribution on or in the porous collagen matrix; and implanting the porous collagen matrix at or near a target tissue site in need of bone repair.

BACKGROUND

Bone is a composite material that is composed of impure hydroxyapatite,collagen and a variety of non-collagenous proteins, as well as embeddedand adherent cells. Due to disease, a congenital defect or an accident,a person may lose or be missing part or all of one or more bones orregions of cartilage in his or her body, and/or have improper growth orformation of bone and/or cartilage.

Mammalian bone tissue is known to contain one or more proteinaceousmaterials that are active during growth and natural bone healing. Thesematerials can induce a developmental cascade of cellular events thatresults in bone formation. Typically, the developmental cascade of boneformation involves chemotaxis of mesenchymal cells, proliferation ofprogenitor cells, differentiation of cartilage, vascular invasion, boneformation, remodeling and marrow differentiation.

When bone is damaged, often bone grafting procedures are performed torepair the damaged bone especially in cases where the damage is complex,poses a significant risk to the patient, and/or fails to heal properly.Bone grafting is also used to help fusion between vertebrae, correctdeformities, or provide structural support for fractures of the spine.In addition to fracture repair, bone grafting is also used to repairdefects in bone caused by birth defects, traumatic injury, or surgeryfor bone cancer.

There are at least three ways in which a bone graft can help repair adefect. The first is called osteogenesis, the formation of new bonewithin the graft. The second is osteoinduction, a process in whichmolecules contained within the graft (e.g., bone morphogenic proteins)convert the patient's cells into cells that are capable of forming bone.The third is osteoconduction, a physical effect by which a matrix oftencontaining graft material acts as a scaffold on which bone and cells inthe recipient are able to form new bone.

The source of bone for grafting can be obtained from bones in thepatient's own body (e.g., hip, skull, ribs, etc.), called autograft, orfrom bone taken from other people that is frozen and stored in tissuebanks, called allograft. The source of bone may also be derived fromanimals of a different species called a xenograft.

Some grafting procedures utilize a variety of natural and syntheticmatrixes with or instead of bone (e.g., collagen, silicone, acrylics,hydroxyapatite, calcium sulfate, ceramics, etc.). To place the matrix atthe bone defect, the surgeon makes an incision in the skin over the bonedefect and shapes the matrix to fit into the defect. As persons ofordinary skill are aware, growth factors (e.g., bone morphogenicprotein-2) may be placed on the matrix in order to spur the patient'sbody to begin the formation of new bone and/or cartilage. These growthfactors act much like a catalyst, encouraging the necessary cells(including, but not limited to, mesenchymal stem cells, osteoblasts, andosteoclasts) to more rapidly migrate into the matrix, which iseventually resorbed via a cell-mediated process and newly formed bone isdeposited at or near the bone defect. In this manner severe fracturesmay be healed, and vertebrae successfully fused.

Sometimes when the surgeon manipulates the matrix to place it in thebone defect, excessive amounts of growth factor (e.g., bone morphogenicprotein) may leak from the matrix, which may reduce a stablemicroenvironment for new bone and/or cartilage growth. It also may causethe matrix to fail to retain its full efficacy over time to maximallypromote bone and/or cartilage growth at a target site. Thus, there is aneed to develop new osteogenic compositions and methods that improvebone and/or cartilage repair.

SUMMARY

Compositions and methods are provided that improve bone and/or cartilagerepair. Through the use of these osteogenic compositions, the growth ofbone, cartilage and/or related tissue may be facilitated. In someembodiments, the osteogenic composition allows the user (e.g., surgeon,nurse, assistant, etc.) to see a visibly discernible color (e.g., green,blue, etc.) so that the user can see the application of the growthfactor. In some embodiments, the osteogenic composition is applied to amatrix that is a contrasting color to the composition (e.g., green orblue composition on white or beige matrix) so that the user can see theconsistency of the application of the growth factor on the matrix or seeunwanted leakage of the growth factor from the matrix. In this way, thematrix can maintain its efficacy over time to promote bone growth at atarget site.

In some embodiments, an osteogenic composition is provided forimplantation at or near a target tissue site beneath the skin, theosteogenic composition comprising a growth factor and a coloring agent,wherein the coloring agent imparts color to the growth factor to allowthe user to implant the growth factor at or near the target tissue site.

In some embodiments, an osteogenic composition is provided forimplantation at or near a target tissue site beneath the skin, theosteogenic composition comprising: a growth factor; a coloring agent;and a biodegradable matrix comprising a porous collagen surface, whereinupon mixing the coloring agent with the growth factor, the coloringagent imparts color to the growth factor to allow the user to see thegrowth factor distribution on or in the porous collagen surface.

In some embodiments, a method is provided for accelerating bone repairin a patient in need of such treatment, the method comprising mixingbone morphogenic protein-2 and a coloring agent to form a mixture,wherein the coloring agent imparts color to the mixture; applying themixture to a surface of a porous collagen matrix, wherein the coloringagent allows the user to see bone morphogenic protein-2 distribution onor in the porous collagen matrix; and implanting the porous collagenmatrix at or near a target tissue site in need of bone repair

Additional features and advantages of various embodiments will be setforth in part in the description that follows, and in part will beapparent from the description, or may be learned by practice of variousembodiments. The objectives and other advantages of various embodimentswill be realized and attained by means of the elements and combinationsparticularly pointed out in the description and appended claims.

DETAILED DESCRIPTION

For the purposes of this specification and appended claims, unlessotherwise indicated, all numbers expressing quantities of ingredients,percentages or proportions of materials, reaction conditions, and othernumerical values used in the specification and claims, are to beunderstood as being modified in all instances by the term “about.”Accordingly, unless indicated to the contrary, the numerical parametersset forth in the following specification and attached claims areapproximations that may vary depending upon the desired propertiessought to be obtained by the present application. At the very least, andnot as an attempt to limit the application of the doctrine ofequivalents to the scope of the claims, each numerical parameter shouldat least be construed in light of the number of reported significantdigits and by applying ordinary rounding techniques.

Notwithstanding that the numerical ranges and parameters setting forththe broad scope of the invention are approximations, the numericalvalues are as precise as possible. Any numerical value, however,inherently contains certain errors necessarily resulting from thestandard deviation found in their respective testing measurements.Moreover, all ranges disclosed herein are to be understood to encompassany and all subranges subsumed therein. For example, a range of “1 to10” includes any and all subranges between (and including) the minimumvalue of 1 and the maximum value of 10, that is, any and all subrangeshaving a minimum value of equal to or greater than 1 and a maximum valueof equal to or less than 10, e.g., 5.5 to 10.

Additionally, unless defined otherwise or apparent from context, alltechnical and scientific terms used herein have the same meanings ascommonly understood by one of ordinary skill in the art to which thisinvention belongs.

Unless explicitly stated or apparent from context, the following termsor phrases have the definitions provided below:

Definitions

It is noted that, as used in this specification and the appended claims,the singular forms “a,” “an,” and “the,” include plural referents unlessexpressly and unequivocally limited to one referent. Thus, for example,reference to “a matrix” includes one, two, three or more matrices.

The term “biodegradable” includes that all or parts of the matrix willdegrade over time by the action of enzymes, by hydrolytic action and/orby other similar mechanisms in the human body. In various embodiments,“biodegradable” includes that a matrix (e.g., microparticle,microsphere, etc.) can break down or degrade within the body tonon-toxic components after or while a therapeutic agent has been or isbeing released. By “bioerodible” it is meant that the matrix will erodeor degrade over time due, at least in part, to contact with substancesfound in the surrounding tissue, fluids or by cellular action. By“bioabsorbable” or “bioresorbable” it is meant that the osteogeniccomposition will be broken down and absorbed within the human body, forexample, by a cell or tissue. “Biocompatible” means that the matrix willnot cause substantial tissue irritation or necrosis at the target tissuesite.

The term “mammal” refers to organisms from the taxonomy class“mammalian,” including but not limited to humans, other primates such aschimpanzees, apes, orangutans and monkeys, rats, mice, cats, dogs, cows,horses, etc.

The term “target tissue site” is intended to mean the location of thetissue to be treated. Typically the placement site of the matrix will bethe same as the target site to provide for optimal targeted drugdelivery. However, the present application also contemplates positioningthe matrix at a placement site at or near the target site such that thetherapeutic agent can be delivered to the surrounding vasculature, whichcarries the agent to the desired nearby target site. As used herein, theterm “at or near” includes embodiments where the placement site andtarget site are within close proximity.

A “therapeutically effective amount” or “effective amount” is such thatwhen administered, the drug results in alteration of the biologicalactivity, such as, for example, promotion of bone, cartilage and/orother tissue (e.g., vascular tissue) growth, inhibition of inflammation,reduction or alleviation of pain, improvement in the condition throughinhibition of an immunologic response, etc. The dosage administered to apatient can be as single or multiple doses depending upon a variety offactors, including the drug's administered pharmacokinetic properties,the route of administration, patient conditions and characteristics(sex, age, body weight, health, size, etc.), extent of symptoms,concurrent treatments, frequency of treatment and the effect desired. Insome embodiments the formulation is designed for immediate release. Inother embodiments the formulation is designed for sustained release. Inother embodiments, the formulation comprises one or more immediaterelease surfaces and one or more sustained release surfaces. In someembodiments, the osteogenic composition comprises an effective amount ofa growth factor.

The phrase “immediate release” is used herein to refer to one or moretherapeutic agent(s) that is introduced into the body and that isallowed to dissolve in or become absorbed at the location to which it isadministered, with no intention of delaying or prolonging thedissolution or absorption of the drug.

The phrases “sustained release” and “sustain release” (also referred toas extended release or controlled release) are used herein to refer toone or more therapeutic agent(s) that is introduced into the body of ahuman or other mammal and continuously or continually releases a streamof one or more therapeutic agents over a predetermined time period andat a therapeutic level sufficient to achieve a desired therapeuticeffect throughout the predetermined time period. Reference to acontinuous or continual release stream is intended to encompass releasethat occurs as the result of biodegradation in vivo of the drug bead,foam and/or component thereof, or as the result of metabolictransformation or dissolution of the therapeutic agent(s) or conjugatesof therapeutic agent(s).

The “matrix” of the present application is utilized as a scaffold forbone and/or cartilage repair, regeneration, and/or augmentation.Typically, the matrix provides a 3-D matrix of interconnecting pores,which acts as a pliant scaffold for cell migration. The morphology ofthe matrix guides cell migration and cells are able to migrate into orover the matrix, respectively. The cells then are able to proliferateand synthesize new tissue and form bone and/or cartilage.

The terms “treating” and “treatment” when used in connection with adisease or condition refer to executing a protocol that may includeosteochondral repair procedure, administering one or more drugs to apatient (human or other mammal), in an effort to alleviate signs orsymptoms of the disease or condition or immunological response.Alleviation can occur prior to signs or symptoms of the disease orcondition appearing, as well as after their appearance. Thus, treatingor treatment includes preventing or prevention of disease or undesirablecondition. In addition, treating, treatment, preventing or prevention donot require complete alleviation of signs or symptoms, does not requirea cure, and specifically includes protocols that have only a marginaleffect on the patient. In some embodiments, the osteogenic compositioncan be used to treat subchondral, osteochondral, hyaline cartilageand/or condyle defects.

The term “subchondral” includes an area underlying joint cartilage. Theterm “subchondral bone” includes a very dense, but thin layer of bonejust below a zone of cartilage and above the cancellous or trabecularbone that forms the bulk of the bone structure of the limb.“Osteochondral” includes a combined area of cartilage and bone where alesion or lesions can occur. “Osteochondral defect” includes a lesionwhich is a composite lesion of cartilage and subchondral bone. “Hyalinecartilage” includes cartilage containing groups of isogenouschondrocytes located within lacunae cavities which are scatteredthroughout an extracellular collagen matrix. A “condyle” includes arounded articular surface of the extremity of a bone.

The phrase “osteogenic composition” refers to a composition thatcomprises a substance that promotes bone growth. In some embodiments,osteogenic compositions as described herein can be delivered to othersurgical sites, particularly sites at which bone growth is desired.These include, for instance, the repair of spine (e.g., vertebraefusion) cranial defects, iliac crest back-filling, acetabular defects,in the repair of tibial plateau, long bone defects, spinal site defectsor the like. Such methods can be used to treat major or minor defects inthese or other bones caused by trauma (including open and closedfractures), disease, or congenital defects, for example.

The term “carrier” includes a diluent, adjuvant, buffer, excipient, orvehicle with which a composition can be administered. Carriers caninclude sterile liquids, such as, for example, water and oils, includingoils of petroleum, animal, vegetable or synthetic origin, such as, forexample, peanut oil, soybean oil, mineral oil, sesame oil, or the like.The growth factor may include a carrier.

The term “excipient” includes a non-therapeutic agent added to apharmaceutical composition to provide a desired consistency orstabilizing effect. Excipients for parenteral formulations, include, forexample, oils (e.g., canola, cottonseed, peanut, safflower, sesame,soybean), fatty acids and salts and esters thereof (e.g., oleic acid,stearic acid, palmitic acid), alcohols (e.g., ethanol, benzyl alcohol),polyalcohols (e.g., glycerol, propylene glycols and polyethyleneglycols, e.g., PEG 3350), polysorbates (e.g., polysorbate 20,polysorbate 80), gelatin, albumin (e.g., human serum albumin), salts(e.g., sodium chloride), succinic acid and salts thereof (e.g., sodiumsuccinate), amino acids and salts thereof (e.g., alanine, histidine,glycine, arginine, lysine), acetic acid or a salt or ester thereof(e.g., sodium acetate, ammonium acetate), citric acid and salts thereof(e.g., sodium citrate), benzoic acid and salts thereof, phosphoric acidand salts thereof (e.g., monobasic sodium phosphate, dibasic sodiumphosphate), lactic acid and salts thereof, polylactic acid, glutamicacid and salts thereof (e.g., sodium glutamate), calcium and saltsthereof (e.g., CaCl₂, calcium acetate), phenol, sugars (e.g., glucose,sucrose, lactose, maltose, trehalose), erythritol, arabitol, isomalt,lactitol, maltitol, mannitol, sorbitol, xylitol, nonionic surfactants(e.g., TWEEN 20, TWEEN 80), ionic surfactants (e.g., sodium dodecylsulfate), chlorobutanol, DMSO, sodium hydroxide, glycerin, m-cresol,imidazole, protamine, zinc and salts thereof (e.g, zinc sulfate),thimerosal, methylparaben, propylparaben, carboxymethylcellulose,chlorobutanol, and heparin, The growth factor may include an excipient.

The term “lyophilized” or “freeze-dried” includes a state of a substancethat has been subjected to a drying procedure such as lyophilization,where at least 50% of moisture has been removed. The growth factor maybe lyophilized or freeze-dried.

A “preservative” includes a bacteriostatic, bacteriocidal, fungistaticor fungicidal compound that is generally added to formulations to retardor eliminate growth of bacteria or other contaminating microorganisms inthe formulations. Preservatives include, for example, benzyl alcohol,phenol, benzalkonium chloride, m-cresol, thimerosol, chlorobutanol,methylparaben, propylparaben and the like. Other examples ofpharmaceutically acceptable preservatives can be found in the USP. Thegrowth factor may have preservatives or be preservative free.

Reference will now be made in detail to certain embodiments of theinvention. While the invention will be described in conjunction with theillustrated embodiments, it will be understood that they are notintended to limit the invention to those embodiments. On the contrary,the invention is intended to cover all alternatives, modifications, andequivalents that may be included within the invention as defined by theappended claims.

Compositions and methods are provided that improve bone and/or cartilagerepair. Through the use of these osteogenic compositions, the growth ofbone, cartilage and/or related tissue may be facilitated. In someembodiments, the osteogenic composition allows the user (e.g., surgeon,nurse, assistant, etc.) to see a visibly discernible color (e.g., green,blue, etc.) so that the user can see the application of the growthfactor. In some embodiments, the osteogenic composition is applied to amatrix that is a contrasting color to the composition (e.g., green orblue composition on white or beige matrix) so that the user can see theconsistency of the application of the growth factor on the matrix or seeunwanted leakage of the growth factor from the matrix. In this way, thematrix can maintain its efficacy over time to promote bone growth at atarget site.

The headings below are not meant to limit the disclosure in any way;embodiments under any one heading may be used in conjunction withembodiments under any other heading. Matrix

The matrix provides a tissue scaffold for the cells to guide the processof tissue formation in vivo in three dimensions. The morphology of thematrix guides cell migration and cells are able to migrate into or overthe scaffold. The cells then are able to proliferate and synthesize newtissue and form bone and/or cartilage. In some embodiments, one or moretissue matrices are stacked on one another.

In some embodiments, the matrix comprises a plurality of pores. In someembodiments, at least 10% of the pores are between about 10 micrometersand about 500 micrometers at their widest points. In some embodiments,at least 20% of the pores are between about 50 micrometers and about 150micrometers at their widest points. In some embodiments, at least 30% ofthe pores are between about 30 micrometers and about 70 micrometers attheir widest points. In some embodiments, at least 50% of the pores arebetween about 10 micrometers and about 500 micrometers at their widestpoints. In some embodiments, at least 90% of the pores are between about50 micrometers and about 150 micrometers at their widest points. In someembodiments, at least 95% of the pores are between about 100 micrometersand about 250 micrometers at their widest points. In some embodiments,100% of the pores are between about 10 micrometers and about 300micrometers at their widest points.

In some embodiments, the matrix has a porosity of at least about 30%, atleast about 50%, at least about 60%, at least about 70%, at least about90%. The pore may support ingrowth of cells, formation or remodeling ofbone, cartilage and/or vascular tissue.

The matrix may comprise natural and/or synthetic material. For example,the tissue scaffold may comprise poly (alpha-hydroxy acids), poly(lactide-co-glycolide) (PLGA), polylactide (PLA), polyglycolide (PG),polyethylene glycol (PEG) conjugates of poly (alpha-hydroxy acids),polyorthoesters (POE), polyaspirins, polyphosphagenes, collagen,hydrolyzed collagen, gelatin, hydrolyzed gelatin, fractions ofhydrolyzed gelatin, elastin, starch, pre-gelatinized starch, hyaluronicacid, chitosan, alginate, albumin, fibrin, vitamin E analogs, such asalpha tocopheryl acetate, d-alpha tocopheryl succinate, D,L-lactide, orL-lactide,,-caprolactone, dextrans, vinylpyrrolidone, polyvinyl alcohol(PVA), PVA-g-PLGA, PEGT-PBT copolymer (polyactive), methacrylates, poly(N-isopropylacrylamide), PEO-PPO-PEO (pluronics), PEO-PPO-PAAcopolymers, PLGA-PEO-PLGA, PEG-PLG, PLA-PLGA, poloxamer 407,PEG-PLGA-PEG triblock copolymers, SAIB (sucrose acetate isobutyrate),polydioxanone, methylmethacrylate (MMA), MMA and N-vinylpyyrolidone,polyamide, oxycellulose, copolymer of glycolic acid and trimethylenecarbonate, polyesteramides, polyetheretherketone,polymethylmethacrylate, silicone, hyaluronic acid, chitosan, orcombinations thereof.

In some embodiments, the matrix may comprise a resorbable ceramic (e.g.,hydroxyapatite, tricalcium phosphate, bioglasses, calcium sulfate, etc.)tyrosine-derived polycarbonate poly (DTE-co-DT carbonate), in which thependant group via the tyrosine—an amino acid—is either an ethyl ester(DTE) or free carboxylate (DT) or combinations thereof.

In some embodiments, the matrix comprises collagen. Exemplary collagensinclude human or non-human (bovine, ovine, and/or porcine), as well asrecombinant collagen or combinations thereof. Examples of suitablecollagen include, but are not limited to, human collagen type I, humancollagen type II, human collagen type III, human collagen type IV, humancollagen type V, human collagen type VI, human collagen type VII, humancollagen type VIII, human collagen type IX, human collagen type X, humancollagen type XI, human collagen type XII, human collagen type XIII,human collagen type XIV, human collagen type XV, human collagen typeXVI, human collagen type XVII, human collagen type XVIII, human collagentype XIX, human collagen type XXI, human collagen type XXII, humancollagen type XXIII, human collagen type XXIV, human collagen type XXV,human collagen type XXVI, human collagen type XXVII, and human collagentype XXVIII, or combinations thereof. Collagen further may comprisehetero- and homo-trimers of any of the above-recited collagen types. Insome embodiments, the collagen comprises hetero- or homo-trimers ofhuman collagen type I, human collagen type II, human collagen type III,or combinations thereof.

In some embodiments, the matrix comprises collagen-containingbiomaterials from the implant market which, when placed in a bonedefect, provide scaffolding around which the patient's new bone willgrow, gradually replacing the carrier matrix as the target site heals.Examples of suitable carrier matrices may include, but are not limitedto, the MasterGraft® Matrix produced by Medtronic Sofamor Danek, Inc.,Memphis, Tenn.; MasterGraft® Putty produced by Medtronic Sofamor Danek,Inc., Memphis, Tenn.; Absorbable Collagen Sponge (“ACS”) produced byIntegra LifeSciences Corporation, Plainsboro, N.J.; bovine skin collagenfibers coated with hydroxyapatite, e.g. Healos®. marketed by Johnson &Johnson, USA; collagen sponges, e.g. Hemostagene® marketed by Coletica SA, France, or e.g. Helisat® marketed by Integra Life Sciences Inc., USA;and Collagraft® Bone Graft Matrix produced by Zimmer Holdings, Inc.,Warsaw, Ind.

In some embodiments, the embodiments the matrix may comprise particlesof bone-derived materials. The bone-derived material may include one ormore of non-demineralized bone particles, demineralized bone particles,lightly demineralized bone particles, and/or deorganified boneparticles.

In some embodiments, the matrix may be seeded with harvested bone cellsand/or bone tissue, such as for example, cortical bone, autogenous bone,allogenic bones and/or xenogenic bone. In some embodiments, the matrixmay be seeded with harvested cartilage cells and/or cartilage tissue(e.g., autogenous, allogenic, and/or xenogenic cartilage tissue). Forexample, before insertion into the target tissue site, the matrix can bewetted with the graft bone tissue/cells, usually with bone tissue/cellsaspirated from the patient, at a ratio of about 3:1, 2:1, 1:1, 1:3 or1:2 by volume. The bone tissue/cells are permitted to soak into thematrix provided, and the matrix may be kneaded by hand or machine,thereby obtaining a pliable consistency that may subsequently be packedinto the bone defect. In some embodiments, the matrix provides amalleable, non-water soluble carrier that permits accurate placement andretention at the implantation site.

The matrix may contain an inorganic material, such as an inorganicceramic and/or bone substitute material. Exemplary inorganic materialsor bone substitute materials include but are not limited to aragonite,dahlite, calcite, amorphous calcium carbonate, vaterite, weddellite,whewellite, struvite, urate, ferrihydrate, francolite, monohydrocalcite,magnetite, goethite, dentin, calcium carbonate, calcium sulfate, calciumphosphosilicate, sodium phosphate, calcium aluminate, calcium phosphate,hydroxyapatite, alpha-tricalcium phosphate, dicalcium phosphate,β-tricalcium phosphate, tetracalcium phosphate, amorphous calciumphosphate, octacalcium phosphate, BIOGLASSTM, fluoroapatite,chlorapatite, magnesium-substituted tricalcium phosphate, carbonatehydroxyapatite, substituted forms of hydroxyapatite (e.g.,hydroxyapatite derived from bone may be substituted with other ions suchas fluoride, chloride, magnesium sodium, potassium, etc.), orcombinations or derivatives thereof.

In some embodiments, tissue will infiltrate the matrix to a degree ofabout at least 50 percent within about 1 month to about 6 months afterimplantation of the matrix. In some embodiments, about 75 percent of thematrix will be infiltrated by tissue within about 2-3 months afterimplantation of the matrix. In some embodiments, the matrix will besubstantially, e.g., about 90 percent or more, submerged in or envelopedby tissue within about 6 months after implantation of the matrix. Insome embodiments, the matrix will be completely submerged in orenveloped by tissue within about 9-12 months after implantation.

In some embodiments, the matrix has a thickness of from 0.25 mm to 5 mm,or from about 0.4 mm to about 2 mm, or 0.4 mm to about 1 mm. Clearly,different bone defects (e.g., osteochondral defects) may requiredifferent matrix thicknesses.

In some embodiments, the matrix has a density of between about 1.6g/cm³, and about 0.05 g/cm³. In some embodiments, the matrix has adensity of between about 1.1 g/cm³, and about 0.07 g/cm³. For example,the density may be less than about 1 g/cm³, less than about 0.7 g/cm³,less than about 0.6 g/cm³, less than about 0.5 g/cm³, less than about0.4 g/cm³, less than about 0.3 g/cm³, less than about 0.2 g/cm³, or lessthan about 0.1 g/cm³.

The shape of the matrix may be tailored to the site at which it is to besituated. For example, it may be in the shape of a morsel, a plug, apin, a peg, a cylinder, a block, a wedge, a sheet, etc.

In some embodiments, the diameter or diagonal of the matrix can rangefrom 1 mm to 50 mm. In some embodiments, the diameter or diagonal of thematrix can range from 1 mm to 30 mm, or 5 mm to 10 mm which is smallenough to fit through an endoscopic cannula, but large enough tominimize the number of matrices needed to fill a large the bone defect(e.g., osteochondral defect).

In some embodiments, the matrix may be made by injection molding,compression molding, blow molding, thermoforming, die pressing, slipcasting, electrochemical machining, laser cutting, water-jet machining,electrophoretic deposition, powder injection molding, sand casting,shell mold casting, lost tissue scaffold casting, plaster-mold casting,ceramic-mold casting, investment casting, vacuum casting, permanent-moldcasting, slush casting, pressure casting, die casting, centrifugalcasting, squeeze casting, rolling, forging, swaging, extrusion,shearing, spinning, powder metallurgy compaction or combinations thereof

In some embodiments, a therapeutic agent (including one or more growthfactors) may be disposed on or in the matrix by hand, electrospraying,ionization spraying or impregnating, vibratory dispersion (includingsonication), nozzle spraying, compressed-air-assisted spraying, brushingand/or pouring. For example, a growth factor such as rhBMP-2 may bedisposed on or in the matrix.

In some embodiments, the matrix may comprise sterile and/or preservativefree material.

The matrix can be implanted by hand or machine in procedures such as forexample, laparoscopic, arthroscopic, neuroendoscopic, endoscopic,rectoscopic procedures or the like.

Growth Factors

In some embodiments, a growth factor and/or therapeutic agent may bedisposed on or in the matrix by hand, electrospraying, ionizationspraying or impregnating, vibratory dispersion (including sonication),nozzle spraying, compressed-air-assisted spraying, brushing and/orpouring. For example, a growth factor such as rhBMP-2 may be disposed onor in the biodegradable carrier by the surgeon before the biodegradablematrix is administered or it may be available from the manufacturerbeforehand.

The biodegradable matrix may comprise at least one growth factor. Thesegrowth factors include osteoinductive agents (e.g., agents that causenew bone growth in an area where there was none) and/or osteoconductiveagents (e.g., agents that cause ingrowth of cells into and/or throughthe matrix). Osteoinductive agents can be polypeptides orpolynucleotides compositions. Polynucleotide compositions of theosteoinductive agents include, but are not limited to, isolated BoneMorphogenic Protein (BMP), Vascular Endothelial Growth Factor (VEGF),Connective Tissue Growth Factor (CTGF), Osteoprotegerin, GrowthDifferentiation Factors (GDFs), Cartilage Derived Morphogenic Proteins(CDMPs), Lim Mineralization Proteins (LMPs), Platelet derived growthfactor, (PDGF or rhPDGF), Insulin-like growth factor (IGF) orTransforming Growth Factor beta (TGF-beta) polynucleotides.Polynucleotide compositions of the osteoinductive agents include, butare not limited to, gene therapy vectors harboring polynucleotidesencoding the osteoinductive polypeptide of interest. Gene therapymethods often utilize a polynucleotide, which codes for theosteoinductive polypeptide operatively linked or associated to apromoter or any other genetic elements necessary for the expression ofthe osteoinductive polypeptide by the target tissue. Such gene therapyand delivery techniques are known in the art (see, for example,International Publication No. WO90/11092, the disclosure of which isherein incorporated by reference in its entirety). Suitable gene therapyvectors include, but are not limited to, gene therapy vectors that donot integrate into the host genome. Alternatively, suitable gene therapyvectors include, but are not limited to, gene therapy vectors thatintegrate into the host genome.

In some embodiments, the polynucleotide is delivered in plasmidformulations. Plasmid DNA or RNA formulations refer to polynucleotidesequences encoding osteoinductive polypeptides that are free from anydelivery vehicle that acts to assist, promote or facilitate entry intothe cell, including viral sequences, viral particles, liposomeformulations, lipofectin, precipitating agents or the like. Optionally,gene therapy compositions can be delivered in liposome formulations andlipofectin formulations, which can be prepared by methods well known tothose skilled in the art. General methods are described, for example, inU.S. Pat. Nos. 5,593,972, 5,589,466, and 5,580,859, the disclosures ofwhich are herein incorporated by reference in their entireties.

Gene therapy vectors further comprise suitable adenoviral vectorsincluding, but not limited to for example, those described in U.S. Pat.No. 5,652,224, which is herein incorporated by reference.

Polypeptide compositions of the isolated osteoinductive agents include,but are not limited to, isolated Bone Morphogenic Protein (BMP),Vascular Endothelial Growth Factor (VEGF), Connective Tissue GrowthFactor (CTGF), Osteoprotegerin, Growth Differentiation Factors (GDFs),Cartilage Derived Morphogenic Proteins (CDMPs), Lim MineralizationProteins (LMPs), Platelet derived growth factor, (PDGF or rhPDGF),Insulin-like growth factor (IGF) or Transforming Growth Factor beta(TGF-beta707) polypeptides. Polypeptide compositions of theosteoinductive agents include, but are not limited to, full lengthproteins, fragments or variants thereof.

Variants of the isolated osteoinductive agents include, but are notlimited to, polypeptide variants that are designed to increase theduration of activity of the osteoinductive agent in vivo. Typically,variant osteoinductive agents include, but are not limited to, fulllength proteins or fragments thereof that are conjugated to polyethyleneglycol (PEG) moieties to increase their half-life in vivo (also known aspegylation). Methods of pegylating polypeptides are well known in theart (See, e.g., U.S. Pat. No. 6,552,170 and European Pat. No. 0,401,384as examples of methods of generating pegylated polypeptides). In someembodiments, the isolated osteoinductive agent(s) are provided as fusionproteins. In one embodiment, the osteoinductive agent(s) are availableas fusion proteins with the Fc portion of human IgG. In anotherembodiment, the osteoinductive agent(s) are available as hetero- orhomodimers or multimers. Examples of some fusion proteins include, butare not limited to, ligand fusions between mature osteoinductivepolypeptides and the Fc portion of human Immunoglobulin G (IgG). Methodsof making fusion proteins and constructs encoding the same are wellknown in the art.

Isolated osteoinductive agents that are included within a matrix aretypically sterile. In a non-limiting method, sterility is readilyaccomplished for example by filtration through sterile filtrationmembranes (e.g., 0.2 micron membranes or filters). In one embodiment,the isolated osteoinductive agents include one or more members of thefamily of Bone Morphogenic Proteins (“BMPs”). BMPs are a class ofproteins thought to have osteoinductive or growth-promoting activitieson endogenous bone tissue, or function as pro-collagen precursors. Knownmembers of the BMP family include, but are not limited to, BMP-1, BMP-2,BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8, BMP-9, BMP-10, BMP-11, BMP-12,BMP-13, BMP-15, BMP-16, BMP-17, BMP-18 as well as polynucleotides orpolypeptides thereof, as well as mature polypeptides or polynucleotidesencoding the same.

BMPs utilized as osteoinductive agents comprise one or more of BMP-1;BMP-2; BMP-3; BMP-4; BMP-5; BMP-6; BMP-7; BMP-8; BMP-9; BMP-10; BMP-11;BMP-12; BMP-13; BMP-15; BMP-16; BMP-17; or BMP-18; as well as anycombination of one or more of these BMPs, including full length BMPs orfragments thereof, or combinations thereof, either as polypeptides orpolynucleotides encoding the polypeptide fragments of all of the recitedBMPs. The isolated BMP osteoinductive agents may be administered aspolynucleotides, polypeptides, full length protein or combinationsthereof.

In another embodiment, isolated osteoinductive agents includeosteoclastogenesis inhibitors to inhibit bone resorption of the bonetissue surrounding the site of implantation by osteoclasts. Osteoclastand osteoclastogenesis inhibitors include, but are not limited to,osteoprotegerin polynucleotides or polypeptides, as well as matureosteoprotegerin proteins, polypeptides or polynucleotides encoding thesame. Osteoprotegerin is a member of the TNF-receptor superfamily and isan osteoblast-secreted decoy receptor that functions as a negativeregulator of bone resorption. This protein specifically binds to itsligand, osteoprotegerin ligand (TNFSF11/OPGL), both of which are keyextracellular regulators of osteoclast development.

Osteoclastogenesis inhibitors further include, but are not limited to,chemical compounds such as bisphosphonate, 5-lipoxygenase inhibitorssuch as those described in U.S. Pat. Nos. 5,534,524 and 6,455,541 (thecontents of which are herein incorporated by reference in theirentireties), heterocyclic compounds such as those described in U.S. Pat.No. 5,658,935 (herein incorporated by reference in its entirety),2,4-dioxoimidazolidine and imidazolidine derivative compounds such asthose described in U.S. Pat. Nos. 5,397,796 and 5,554,594 (the contentsof which are herein incorporated by reference in their entireties),sulfonamide derivatives such as those described in U.S. Pat. No.6,313,119 (herein incorporated by reference in its entirety), oracylguanidine compounds such as those described in U.S. Pat. No.6,492,356 (herein incorporated by reference in its entirety).

In another embodiment, isolated osteoinductive agents include one ormore members of the family of Connective Tissue Growth Factors(“CTGFs”). CTGFs are a class of proteins thought to havegrowth-promoting activities on connective tissues. Known members of theCTGF family include, but are not limited to, CTGF-1, CTGF-2, CTGF-4polynucleotides or polypeptides thereof, as well as mature proteins,polypeptides or polynucleotides encoding the same.

In another embodiment, isolated osteoinductive agents include one ormore members of the family of Vascular Endothelial Growth Factors(“VEGFs”). VEGFs are a class of proteins thought to havegrowth-promoting activities on vascular tissues. Known members of theVEGF family include, but are not limited to, VEGF-A, VEGF-B, VEGF-C,VEGF-D, VEGF-E or polynucleotides or polypeptides thereof, as well asmature VEGF-A, proteins, polypeptides or polynucleotides encoding thesame.

In another embodiment, isolated osteoinductive agents include one ormore members of the family of Transforming Growth Factor-beta(“TGFbetas”). TGF-betas are a class of proteins thought to havegrowth-promoting activities on a range of tissues, including connectivetissues. Known members of the TGF-beta family include, but are notlimited to, TGF-beta-1, TGF-beta-2, TGF-beta-3, polynucleotides orpolypeptides thereof, as well as mature protein, polypeptides orpolynucleotides encoding the same.

In another embodiment, isolated osteoinductive agents include one ormore Growth Differentiation Factors (“GDFs”). Known GDFs include, butare not limited to, GDF-1, GDF-2, GDF-3, GDF-7, GDF-10, GDF-11, andGDF-15. For example, GDFs useful as isolated osteoinductive agentsinclude, but are not limited to, the following GDFs: GDF-1polynucleotides or polypeptides corresponding to GenBank AccessionNumbers M62302, AAA58501, and AAB94786, as well as mature GDF-1polypeptides or polynucleotides encoding the same. GDF-2 polynucleotidesor polypeptides corresponding to GenBank Accession Numbers BC069643,BC074921, Q9UK05, AAH69643, or AAH74921, as well as mature GDF-2polypeptides or polynucleotides encoding the same. GDF-3 polynucleotidesor polypeptides corresponding to GenBank Accession Numbers AF263538,BCO30959, AAF91389, AAQ89234, or Q9NR23, as well as mature GDF-3polypeptides or polynucleotides encoding the same. GDF-7 polynucleotidesor polypeptides corresponding to GenBank Accession Numbers AB158468,AF522369, AAP97720, or Q7Z4P5, as well as mature GDF-7 polypeptides orpolynucleotides encoding the same. GDF-10 polynucleotides orpolypeptides corresponding to GenBank Accession Numbers BCO28237 orAAH28237, as well as mature GDF-10 polypeptides or polynucleotidesencoding the same.

GDF-11 polynucleotides or polypeptides corresponding to GenBankAccession Numbers AF100907, NP_(—)005802 or 095390, as well as matureGDF-11 polypeptides or polynucleotides encoding the same. GDF-15polynucleotides or polypeptides corresponding to GenBank AccessionNumbers BC008962, BC000529, AAH00529, or NP 004855, as well as matureGDF-15 polypeptides or polynucleotides encoding the same.

In another embodiment, isolated osteoinductive agents include CartilageDerived Morphogenic Protein (CDMP) and Lim Mineralization Protein (LMP)polynucleotides or polypeptides. Known CDMPs and LMPs include, but arenot limited to, CDMP-1, CDMP-2, LMP-1, LMP-2, or LMP-3.

CDMPs and LMPs useful as isolated osteoinductive agents include, but arenot limited to, the following CDMPs and LMPs: CDMP-1 polynucleotides andpolypeptides corresponding to GenBank Accession Numbers NM_000557,U13660, NP_000548 or P43026, as well as mature CDMP-1 polypeptides orpolynucleotides encoding the same. CDMP-2 polypeptides corresponding toGenBank Accession Numbers or P55106, as well as mature CDMP-2polypeptides. LMP-1 polynucleotides or polypeptides corresponding toGenBank Accession Numbers AF345904 or AAK30567, as well as mature LMP-1polypeptides or polynucleotides encoding the same. LMP-2 polynucleotidesor polypeptides corresponding to GenBank Accession Numbers AF345905 orAAK30568, as well as mature LMP-2 polypeptides or polynucleotidesencoding the same. LMP-3 polynucleotides or polypeptides correspondingto GenBank Accession Numbers AF345906 or AAK30569, as well as matureLMP-3 polypeptides or polynucleotides encoding the same.

In another embodiment, isolated osteoinductive agents include one ormore members of any one of the families of Bone Morphogenic Proteins(BMPs), Connective Tissue Growth Factors (CTGFs), Vascular EndothelialGrowth Factors (VEGFs), Osteoprotegerin or any of the otherosteoclastogenesis inhibitors, Growth Differentiation Factors (GDFs),Cartilage Derived Morphogenic Proteins (CDMPs), Lim MineralizationProteins (LMPs), or Transforming Growth Factor-betas (TGF-betas), aswell as mixtures or combinations thereof.

In another embodiment, the one or more isolated osteoinductive agentsuseful in the bioactive formulation are selected from the groupconsisting of BMP-1, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8,BMP-9, BMP-10, BMP-11, BMP-12, BMP-13, BMP-15, BMP-16, BMP-17, BMP-18,or any combination thereof; CTGF-1, CTGF-2, CGTF-3, CTGF-4, or anycombination thereof; VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, or anycombination thereof; GDF-1, GDF-2, GDF-3, GDF-7, GDF-10, GDF-11, GDF-15,or any combination thereof; CDMP-1, CDMP-2, LMP-1, LMP-2, LMP-3, and/orany combination thereof; Osteoprotegerin; TGF-beta-1, TGF-beta-2,TGF-beta-3, or any combination thereof; or any combination of one ormore members of these groups.

In some embodiments, BMP-7 and/or GDF-5 may be used at 1-2mg/cc ofmatrix.

The concentrations of growth factor can be varied based on the desiredlength or degree of osteogenic effects desired. Similarly, one of skillin the art will understand that the duration of sustained release of thegrowth factor can be modified by the manipulation of the compositionscomprising the sustained release formulation, such as for example,modifying the percent of polymers found within a sustained releaseformulation, microencapsulation of the formulation within polymers,including polymers having varying degradation times and characteristics,and layering the formulation in varying thicknesses in one or moredegradable polymers. These sustained release formulations can thereforebe designed to provide customized time release of growth factors thatstimulate the natural healing process.

The growth factor may contain inactive materials such as bufferingagents and pH adjusting agents such as potassium bicarbonate, potassiumcarbonate, potassium hydroxide, sodium acetate, sodium borate, sodiumbicarbonate, sodium carbonate, sodium hydroxide or sodium phosphate;degradation/release modifiers; drug release adjusting agents;emulsifiers; preservatives such as benzalkonium chloride, chlorobutanol,phenylmercuric acetate and phenylmercuric nitrate, sodium bisulfate,sodium bisulfite, sodium thiosulfate, thimerosal, methylparaben,polyvinyl alcohol and phenylethyl alcohol; solubility adjusting agents;stabilizers; and/or cohesion modifiers. In some embodiments, the growthfactor may comprise sterile and/or preservative free material.

These above inactive ingredients may have multi-functional purposesincluding the carrying, stabilizing and controlling the release of thegrowth factor and/or other therapeutic agent(s). The sustained releaseprocess, for example, may be by a solution-diffusion mechanism or it maybe governed by an erosion-sustained process.

In some embodiments, a pharmaceutically acceptable formulationcomprising a growth factor is provided, wherein the formulation is afreeze-dried or lyophilized formulation. Typically, in the freeze-driedor lyophilized formulation an effective amount of a growth factor isprovided. Lyophilized formulations can be reconstituted into solutions,suspensions, emulsions, or any other suitable form for administration oruse. The lyophilized formulation may comprise the coloring agent or theliquid used to reconstitute the growth factor may contain the coloringagent. Lyophilized formulations are typically first prepared as liquids,then frozen and lyophilized. The total liquid volume beforelyophilization can be less, equal to, or more than, the finalreconstituted volume of the lyophilized formulation. The lyophilizationprocess is well known to those of ordinary skill in the art, andtypically includes sublimation of water from a frozen formulation undercontrolled conditions.

Lyophilized formulations can be stored at a wide range of temperatures.Lyophilized formulations may be stored at or below 30° C., for example,refrigerated at 4° C., or at room temperature (e.g., approximately 25°C.).

Lyophilized formulations of the growth factor are typicallyreconstituted for use by addition of an aqueous solution to dissolve thelyophilized formulation. A wide variety of aqueous solutions can be usedto reconstitute a lyophilized formulation. In some embodiments,lyophilized formulations are reconstituted using water. In someembodiments, the coloring agent may be added to the diluent thatreconstitutes the growth factor. In some embodiments, lyophilizedformulations can be reconstituted with a solution containing water(e.g., USP WFI, or water for injection) or bacteriostatic water (e.g.,USP WFI with 0.9% benzyl alcohol). However, solutions comprising buffersand/or excipients and/or one or more pharmaceutically acceptable carriescan also be used. In some embodiments, the solutions do not contain anypreservatives (e.g., are preservative free).

In some embodiments, the lyophilized growth factor (e.g., BMP) can bedisposed in a vial by the manufacturer and then the surgeon can mix thediluent containing the coloring agent with the lyophilized growthfactor. This mixture can then be parenterally administered to the targettissue site. The term “parenteral” as used herein refers to modes ofadministration which bypass the gastrointestinal tract, and include forexample, intramuscular, intraperitoneal, intrasternal, subcutaneous,intra-operatively, intrathecally, intradiskally, peridiskally,epidurally, perispinally, intraarticular or combinations thereof.

In some embodiments, the growth factor (e.g., BMP) is a colorlesssolution and the coloring agent can be added to the colorless solutionso that the user can now see the growth factor's application to thetarget tissue site and/or matrix. In some embodiments, the mixture ofgrowth factor and coloring agent is applied to the matrix and then theuser can view its distribution on the matrix.

The amount of growth factor, e.g., bone morphogenic protein may besufficient to cause bone and/or cartilage growth. In some embodiments,the growth factor is rhBMP-2 and is contained in one or more matrices inan amount of from 1 to 2 mg per cubic centimeter of the biodegradablematrix. In some embodiments, the amount of rhBMP-2 morphogenic proteinis from 2.0 to 2.5 mg per cubic centimeter (cc) of the biodegradablematrix.

In some embodiments, the growth factor is supplied in a liquid carrier(e.g., an aqueous buffered solution). Exemplary aqueous bufferedsolutions include, but are not limited to, TE, HEPES(2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid), MES(2-morpholinoethanesulfonic acid), sodium acetate buffer, sodium citratebuffer, sodium phosphate buffer, a Tris buffer (e.g., Tris-HCL),phosphate buffered saline (PBS), sodium phosphate, potassium phosphate,sodium chloride, potassium chloride, glycerol, calcium chloride or acombination thereof. In various embodiments, the buffer concentrationcan be from about 1 mM to 100 mM. In some embodiments, the BMP-2 isprovided in a vehicle (including a buffer) containing sucrose, glycine,L-glutamic acid, sodium chloride, and/or polysorbate 80.

In some embodiments, upon implantation of the matrix or components thatcontact the matrix (e.g., plugs that are separate from the matrix onimplantation), some compression of the matrix occurs that causes thebuffer from the bone growth factor to leak from the carrier, whichcauses higher concentrations of the growth factor (e.g., 2 mg to 2.5 mgof rhBMP-2 per cc of matrix) to remain on the matrix. This highconcentration of growth factor may lead to local transient boneresorption and excess osteoclast formation and bone breakdown. This mayresult in poor integration of the plug graft with surrounding hosttissue and a failed repair. In some embodiments, localized release ofthe growth factor may cause local irritation to the surrounding tissue.In some embodiments, the leaking of growth factor from the matrix mayreduce a stable microenvironment for new bone and/or cartilage growth.It also may cause the matrix to fail to retain its full efficacy overtime to maximally promote bone growth at a target site.

In some embodiments, some compression may cause the growth factor fromthe upper part of the matrix to migrate to the lower part and thus causea low concentration of growth factor (e.g., 0.05 mg to 1 mg of rhBMP-2per cc of matrix) on the upper part of the matrix to be exposed to themicroenvironment of the defect, which may promote cartilage formation atthe site the matrix is implanted. In some instances, this may bedesirable especially where more cartilage tissue is needed.

In some embodiments, when the matrix is used in conjunction withosteochondral plugs, if compression occurs, the pores of the matrix getclosed and the plug drops from the surface of the cartilage so thesurface of the plug is no longer flush with the surface of thecartilage. This will cause poor tissue growth.

In some embodiments, a method is provided for accelerating bone repairin a patient in need of such treatment, the method comprising mixing agrowth factor (e.g., bone morphogenic protein-2) and a coloring agent toform a mixture, wherein the coloring agent imparts color to the mixture;applying the mixture to a surface of a porous collagen matrix, whereinthe coloring agent allows the user to see the growth factor distributionon or in the porous collagen matrix; and implanting the porous collagenmatrix at or near a target tissue site in need of bone repair. In thisway, the user can see the distribution of the growth factor on thematrix and/or see the matrix is over-compressed by the leakage of thecoloring agent from the matrix.

Coloring Agent

By adding a coloring agent to growth factor, the user can visualize ifexcessive compression of the matrix occurs by looking for growth factorleakage simply by watching for the color. In this way, if a color isvisualized off the matrix, the user can discard the matrix, remove theleakage with a suctioning instrument or, if compression is notexcessive, stop pressing on the matrix.

In some embodiments, by adding a coloring agent to the growth factor,the user can visualize not only compression of the matrix, but alsoapplication of the coloring agent to the matrix. The user can view thematrix and determine if the growth factor has been applied to one ormore surfaces of the matrix or if the growth factor is uniformlydistributed on or in the matrix simply by viewing the color distributionalong the matrix.

In some embodiments, to ease viewing, the coloring agent may have acolor in stark contrast to the matrix. For example, the matrix may bewhite or beige and the coloring agent with growth factor may be green orblue. In this way, the growth factor can easily be seen. In someembodiments, the coloring agent and/or matrix may be a color that is instark contrast to surrounding tissue and/or blood. For example, thecolor red may be avoided if the procedure will be bloody.

It will be understood by those of ordinary skill in the art that somecompression may occur to release the growth factor (e.g., less than 0.75M Pa, or 0.5 M Pa, or 0.25 M Pa of pressure), which may release lessthan 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or 0.5% w/w or w/v of thegrowth factor. The matrix may be in a compressed state, for example,when more than 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or all ofthe growth factor leaks out of the matrix.

Coloring agents that may be used to color the growth factor includecommercially available natural and artificial colorants. One or morecoloring agents are included in growth factor formulation or they can beadded to it at the time of use or by the manufacturer and thenreconstituted at the time of use.

The coloring agent can be in the osteogenic composition in an amount ofbetween about 0.5% and 99%, or between 1% and 90%, or between 10% and70% or between 20% and 50%, or between about 1% and about 20%, orbetween about 0.5% and about 10% (w/w, w/v, or v/v) with respect to thetotal composition (w/w, w/v, v/v) to impart the desired color to thegrowth factor.

In some embodiments, the coloring agent will be separate from the bufferand the growth factor. In some embodiments, the coloring agent and theliquid buffer (e.g., containing sucrose, glycine, L-glutamic acid,sodium chloride, and/or polysorbate 80) will be together in one vial,which can be used to reconstitute lyophilized or freeze dried growthfactor (e.g., rhBMP-2), which is also in a separate vial. Once the twoare mixed, the user can now visualize administration of the growthfactor.

Color is understood to mean any color in the visible spectrum. In someembodiments, the specific color of the coloring agent is transmitted invisible light. In some embodiments, the color of the coloring agent canbe red, yellow, green, blue, violet, or the like. The coloring agent isunderstood to mean a material intended to give a color to a material orcomposition. The coloring agent may comprise a pigment for it to givethe desired color. Pigments are natural or synthetic substances composedof fine particles which, in contrast to dyes, are often insoluble intheir medium of use, the main function of which is to give a coloring.Pigments may be inorganic pigments, organic pigments (e.g., carbon black(C1 77 266) or D & C Red 36), lakes or pearlescent pigments. Lakes aredyes adsorbed on insoluble particles, the combination remainingessentially insoluble in the medium of use. Pearlescent pigments arenatural or synthetic substances which scatter and reflect light to givean iridescent or bright effect. The dyes may include natural organicdyes, such as cochineal carmine (C1 75 470), or synthetic organic dyes,such as haloacid, azo or anthraquinone dyes. Dyes may include inorganicdyes, such as copper sulphate.

Inorganic pigments of metal oxides in particular zirconium, cerium, zincor chromium oxides (C1 77 288), titanium dioxide (C1 77 891), black,yellow, red and brown iron oxides (C1 77 499, C1 77 492 or C1 77 491),manganese violet (C1 77 742), ultramarine blue (C1 77 007), iron blue(C1 77 510), chromium hydrate (C1 77 289), silver powder or aluminiumpowder or combinations thereof may be used.

Lakes are generally composed of metal salts (in particular Al, Zr, Ca orNa) of organic dyes adsorbed on particles, for example of alumina, ofbarium sulphate, of colophony, and the like. Lakes include those knownunder the names: D & C Red 21 (C1 45 380), D & C Orange 5 (C1 45 370), D& C Red 27 (CI 45 410), D & C Orange 10 (C1 45 425), D & C Red 3 (C145430), D & C Red 7 (C1 15 850:1), D & C Red 4 (C1 15 510), D & C Red 33(C1 17 200), D & C Yellow 5 (C1 19 140), D & C Yellow 6 (C1 15 985), D &C Green 5 (C1 61 570), D & C Yellow 10 (C1 77 002), D & C Green 3 (C1 42053), D & C Blue 1 (C1 42 090).

Pearlescent pigments may include bismuth oxychloride or mica coveredwith titanium oxide, with iron oxide or with natural pigments, forexample colored titanium dioxide-coated mica. A combination of one ormore above coloring agents can be used to produce the desired color ofthe formulation.

In some embodiments, the coloring agent maintains its color before,during and after mixing. For example, in some embodiments, the coloringagent does not need certain pH and/or temperature characteristics tooccur to promote a change of color (e.g., pH causing red to blue colorchange, or temperature to cause clear to blue color). Rather, in someembodiments, the coloring agent maintains its color before, during andafter mixing with the growth factor and/or diluent. Thus, in thisembodiment, no special UV light, or detection instrument is needed andthe coloring agent will maintain its color during the period of mixingand administering the growth factor. The coloring agent will be visiblyapparent to the naked eye of a person of normal color vision. This isunlike thermosetting dyes or pH sensitive dyes that change color whencertain pH or temperature is reached.

In some embodiments, the coloring agent (thus the growth factor whenmixed) will be readily visible to the human eye (including a userwearing glasses, face shield and/or contacts). Some exemplary coloringagents are synthetic or natural agents that are FDA approved. Theseinclude D&C violet #2, D&C red #22, D&C red #27, D&C red #28, D&C red#33, D&C orange #4, D&C yellow #10, D&C green #5, D&C green #8, D&C redNo. 6 barium lake, D&C Red No. 7 calcium lake, D&C red No. 7 calciumlake, D&C red No. 7 calcium lake, D&C red No. 27 aluminum lake, D&C redNo. 27 Zr/Al titanium 02 lake, D&C red No. 28 aluminum lake, D&C red No.30 talc lake, D&C yellow No. 10 aluminum lake, FD&C blue 1, FD&C green3, FD&C red 40, FD&C yellow 5, FD&C yellow 6, D&C green 5, D&C green 8,D&C orange 4. D&C orange 5, D&C red 22, D&C red 33D, D&C violet 2,annatto, bismuth oxychloride, carmine carotene-beta, chromium hydroxidegreen, chromium oxide greens, ferric ferrocyanide, henna, iron oxides,manganese violet, mica, titanium dioxide, ultramarines, zinc oxide or acombination thereof.

In some embodiments, the colorant comprises one or more of FD&C blue No.1—brilliant blue FCF, E133 (blue shade), FD&C blue No. 2—indigotine,E132 (dark blue shade), FD&C green No. 3—fast green FCF, E143 (bluishgreen shade), FD&C red No. 40—allura red AC, E129 (red shade), FD&C redNo. 3—erythrosine, E127 (pink shade), FD&C yellow No. 5—tartrazine, E102(yellow shade), FD&C yellow No. 6—sunset yellow FCF, E110 (orangeshade), congo red, indigo, methylene blue, or a combination thereof.

In some embodiments, the coloring agent is approved by the U.S. FDA foruse in or on medical devices. These coloring agents include chromiumoxide greens (blue-green pigment), phthalocyanine green (pigment),7,16-Dichloro-6,15-dihydro-5, 9, 14, 18-anthrazinetetrone or C.I. Vatblue 6 (Vat Dye), 7,16-Dichloro-6,15-dihydro-5, 9, 14,18-anthrazinetetrone or C.I. Vat Green (vat Dye), D&C Blue No. 9(A.C.I., Vat Blue 6 with other additives), FD&C Blue No. 2 (Lake), D&CBlue No. 6 (water soluble dye), D&C Green No. 5 (pigment), D&C Green No.6 (water soluble dye), or a combination thereof.

In some embodiments, the coloring agent may be visible by instrument,such as for example, magnifying glass, microscope, spectrophotometer ata given wavelength.

Typically, the growth factor and coloring agent may be provided to thephysician as two-phase or three-phase system that comprises a liquiddiluent (e.g., WFI and the coloring agent) and growth factor or thediluent, coloring agent and the growth factor separately or the growthfactor and/or coloring agent may be disposed on or in the matrix and thediluent can be applied to the matrix to reconstitute the growth factorand/or coloring agent. The user will be able to see the application ofthe growth factor. The growth factor, and/or coloring agent may be indifferent forms (e.g., powder, liquid, gel form, etc.). These can bemixed in the operating room in a vacuum-mixing chamber (oralternatively, may be mixed without using a vacuum-mixing chamber),distributed on or in the matrix and inserted into the prepared bonecavity.

In some embodiments, the coloring agent does not interfere orsubstantially interfere with the growth factor and/or matrix function.

Additional Therapeutic Agents

The growth factors of the present application may be disposed on or inthe matrix with other therapeutic agents. For example, the growth factormay be disposed on or in the carrier by electrospraying, ionizationspraying or impregnating, vibratory dispersion (including sonication),nozzle spraying, compressed-air-assisted spraying, brushing and/orpouring.

Exemplary therapeutic agents include but are not limited to IL-1inhibitors, such Kineret® (anakinra), which is a recombinant,non-glycosylated form of the human interleukin-1 receptor antagonist(IL-1Ra), or AMG 108, which is a monoclonal antibody that blocks theaction of IL-1. Therapeutic agents also include excitatory amino acidssuch as glutamate and aspartate, antagonists or inhibitors of glutamatebinding to NMDA receptors, AMPA receptors, and/or kainate receptors.Interleukin-1 receptor antagonists, thalidomide (a TNF-α releaseinhibitor), thalidomide analogues (which reduce TNF-α production bymacrophages), quinapril (an inhibitor of angiotensin II, whichupregulates TNF-α), interferons such as IL-11 (which modulate TNF-αreceptor expression), and aurin-tricarboxylic acid (which inhibitsTNF-α), may also be useful as therapeutic agents for reducinginflammation. It is further contemplated that where desirable apegylated form of the above may be used. Examples of still othertherapeutic agents include NF kappa B inhibitors such as antioxidants,such as dilhiocarbamate, and other compounds, such as, for example,sulfasalazine.

Examples of therapeutic agents suitable for use also include, but arenot limited to, an anti-inflammatory agent, analgesic agent, orosteoinductive growth factor or a combination thereof. Anti-inflammatoryagents include, but are not limited to, apazone, celecoxib, diclofenac,diflunisal, enolic acids (piroxicam, meloxicam), etodolac, fenamates(mefenamic acid, meclofenamic acid), gold, ibuprofen, indomethacin,ketoprofen, ketorolac, nabumetone, naproxen, nimesulide, salicylates,sulfasalazine [2-hydroxy-5-[4-[C2-pyridinylamino)sulfonyl]azo]benzoicacid, sulindac, tepoxalin, and tolmetin; as well as antioxidants, suchas dithiocarbamate, steroids, such as cortisol, cortisone,hydrocortisone, fludrocortisone, prednisone, prednisolone,methylprednisolone, triamcinolone, betamethasone, dexamethasone,beclomethasone, fluticasone or a combination thereof.

Suitable analgesic agents include, but are not limited to,acetaminophen, bupivicaine, fluocinolone, lidocaine, opioid analgesicssuch as buprenorphine, butorphanol, dextromoramide, dezocine,dextropropoxyphene, diamorphine, fentanyl, alfentanil, sufentanil,hydrocodone, hydromorphone, ketobemidone, levomethadyl, mepiridine,methadone, morphine, nalbuphine, opium, oxycodone, papaveretum,pentazocine, pethidine, phenoperidine, piritramide, dextropropoxyphene,remifentanil, tilidine, tramadol, codeine, dihydrocodeine, meptazinol,dezocine, eptazocine, flupirtine, amitriptyline, carbamazepine,gabapentin, pregabalin, or a combination thereof. In some embodiments, astatin may be used. Statins include, but is not limited to,atorvastatin, simvastatin, pravastatin, cerivastatin, mevastatin (seeU.S. Pat. No. 3,883,140, the entire disclosure is herein incorporated byreference), velostatin (also called synvinolin; see U.S. Pat. Nos.4,448,784 and 4,450,171 these entire disclosures are herein incorporatedby reference), fluvastatin, lovastatin, rosuvastatin and fluindostatin(Sandoz XU-62-320), dalvastain (EP Appin. Pubin. No. 738510 A2, theentire disclosure is herein incorporated by reference), eptastatin,pitavastatin, or pharmaceutically acceptable salts thereof or acombination thereof. In various embodiments, the statin may comprisemixtures of (+)R and (−)-S enantiomers of the statin. In variousembodiments, the statin may comprise a 1:1 racemic mixture of thestatin.

Kits

The matrix, coloring agent, growth factor and devices to administer theosteogenic composition may be sterilizable. In various embodiments, oneor more components of the osteogenic composition, and/or medical deviceto administer it may be sterilizable by radiation in a terminalsterilization step in the final packaging. Terminal sterilization of aproduct provides greater assurance of sterility than from processes suchas an aseptic process, which require individual product components to besterilized separately and the final package assembled in a sterileenvironment.

Typically, in various embodiments, gamma radiation is used in theterminal sterilization step, which involves utilizing ionizing energyfrom gamma rays that penetrates deeply in the device. Gamma rays arehighly effective in killing microorganisms, they leave no residues norhave sufficient energy to impart radioactivity to the device. Gamma rayscan be employed when the device is in the package and gammasterilization does not require high pressures or vacuum conditions,thus, package seals and other components are not stressed. In addition,gamma radiation eliminates the need for permeable packaging materials.

In some embodiments, the osteogenic composition may be packaged in amoisture resistant package and then terminally sterilized by gammairradiation. In use the surgeon removes the one or all components fromthe sterile package for use.

In various embodiments, electron beam (e-beam) radiation may be used tosterilize one or more components of the device. E-beam radiationcomprises a form of ionizing energy, which is generally characterized bylow penetration and high-dose rates. E-beam irradiation is similar togamma processing in that it alters various chemical and molecular bondson contact, including the reproductive cells of microorganisms. Beamsproduced for e-beam sterilization are concentrated, highly-chargedstreams of electrons generated by the acceleration and conversion ofelectricity.

Other methods may also be used to sterilize the osteogenic compositionand/or one or more components of the device, including, but not limitedto, gas sterilization, such as, for example, with ethylene oxide orsteam sterilization.

In various embodiments, a kit is provided comprising the growth factor,coloring agent, matrix, and/or diluents. The kit may include additionalparts along with the osteogenic composition combined together to be usedto implant the matrix (e.g., sponges, meshes etc.). The kit may includethe matrix in a first compartment. The second compartment may include avial holding the growth factor, diluent and coloring agent and any otherinstruments needed for the localized drug delivery. A third compartmentmay include gloves, drapes, wound dressings and other proceduralsupplies for maintaining sterility of the implanting process, as well asan instruction booklet, which may include a color chart that shows thecolor of the growth factor after reconstitution with the coloring agent.A fourth compartment may include additional needles and/or sutures. Eachtool may be separately packaged in a plastic pouch that is radiationsterilized. A fifth compartment may include an agent for radiographicimaging. A cover of the kit may include illustrations of the implantingprocedure and a clear plastic cover may be placed over the compartmentsto maintain sterility.

It will be apparent to those skilled in the art that variousmodifications and variations can be made to various embodimentsdescribed herein without departing from the spirit or scope of theteachings herein. Thus, it is intended that various embodiments coverother modifications and variations of various embodiments within thescope of the present teachings.

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 17. A method for accelerating bone repair in a patient inneed of such treatment, the method comprising mixing bone morphogenicprotein-2 and a coloring agent to form a mixture, wherein the coloringagent imparts color to the mixture; applying the mixture to a surface ofa porous collagen matrix, wherein the coloring agent allows a user tosee bone morphogenic protein-2 distribution on or in the porous collagenmatrix; and implanting the porous collagen matrix at or near a targettissue site in need of bone repair.
 18. A method for accelerating bonerepair according to claim 17, wherein bone morphogenic protein-2comprises a colorless solution and the coloring agent comprises a dye.19. A method for accelerating bone repair according to claim 17, whereinthe biodegradable matrix releases bone morphogenic protein-2 and thecoloring agent when the matrix is compressed for the user to see.
 20. Amethod for accelerating bone repair according to claim 17, wherein thecoloring agent comprises a color contrasting the color of the matrix.